Observing various events in real time and at high sensitivity in vivo is extremely important for explaining biological phenomena. One means that has drawn attention in recent years is near-infrared fluorescent imaging using the near-infrared light region from 650 to 900 nm, which is a wavelength region suited to observation in vivo. This means is presumed to be attractive because the equipment is very simple, and the safety is high since no radioactive substances are used. The development of near-infrared fluorescent probes to be used in near-infrared fluorescent imaging is therefore an active research topic.
However, the development of fluorescent probes in the near-infrared light region is less adequate than fluorescent probes of the visible light region. This is due to a lack of useful fluorescence control principles in the near-infrared fluorescence region. FRET, one widely used fluorescence control principle, is an effective fluorescence control method in the near-infrared light region as well, and many near-infrared fluorescent probes that utilize it are being developed. In particular, the fact that the utilization of a non-fluorescent quencher as a FRET acceptor can raise the S/N ratio is leading to the development of useful probes.
However, there are also limitations to the development of FRET-type probes since there are no useful quenchers of the near-infrared light region even when FRET is used as a control mechanism. It would therefore be very useful in the evolution of the life sciences to develop quenchers in the near-infrared light region and to develop a variety of near-infrared fluorescent probes by utilizing them.
In the case of rhodamine which has absorption and fluorescence wavelengths in the visible light region, the fluorescence of rhodamine is known to be quenched and the wavelength lengthened by bonding a phenyl group and a derivative thereof to the nitrogen atoms at positions 3 and 6 (Patent Reference 1). Such non-fluorescent xanthene-type compounds are widely used as quenchers. However, their absorption wavelength is not reached until the second half of 700 nm. They are consequently unsuitable as quenchers of fluorophores such as Cy7, Cy7.5, and ICG which are common near-infrared fluorophores.
Thus, near-infrared quenchers that can be used as the scaffold structure of fluorescent probes in the near-infrared light region have not yet been developed.